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The polymerase chain reaction (PCR) is a powerful method for amplifying nucleic acid sequences. Various disclosures involving this technique are found in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; 4,965,188; and 5,512,462, each of which is incorporated herein by reference. In a simple form, PCR is an in vitro technique for the enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to complementary nucleic acid strands and flank a region that is to be amplified in a target DNA. A series of reaction steps of 1) template denaturation, 2) primer annealing, and 3) extension of annealed primers by DNA polymerase, results in the geometric accumulation of a specific fragment whose termini are defined by the 5xe2x80x2 ends of the primers. As is well known, PCR is capable of selective enrichment of specific DNA sequences by a factor of 109.
PCR has been applied widely in molecular biology for sequencing, genome mapping and forensics. However, despite such wide-spread use, amplifying long stretches of DNA, particularly genomic DNA, is difficult. Many protocols for long range PCR exist; however, reaction conditions are usually optimized for amplifying specific target regions of interest. Applying the same xe2x80x9coptimizedxe2x80x9d reaction conditions to amplify a different target region may not result in a detectable amplification product.
In light of the above limitations, there is a need in the art for methods capable of amplifying nucleic acid sequences. The resulting methods may be used in some embodiments to amplify mammalian target sequences across the genome to facilitate genotyping studies, and for other applications in the art of molecular biology.
The presently claimed invention provides methods for amplifying a DNA target sequence. One embodiment of the present invention provides robust methods for amplification of target sequences. In a first aspect of the invention, a method for designing primer pairs for the amplification reaction is provided. In a further aspect of the invention, reagents and cycling parameters for the amplification reaction are provided.
Thus, the present invention provides a method for designing primer pairs for amplifying a target sequence, comprising the steps of: choosing a reference sequence; removing at least selected repeat regions in the reference sequence to yield removed and unremoved reference sequence; selecting primer sequences from the unremoved reference sequence according to two or more parameters including primer length and primer melting temperature yield a set of primers; evaluating the set of primers for extent of coverage and overlap of the reference sequence; and selecting a subset of primer pairs having reduced overlap from the set of primers.
In addition, the present invention provides a method for amplifying a target sequence, comprising the steps of: mixing a reaction cocktail comprising deoxynucleotide triphosphates, target DNA, a divalent cation, DNA polymerase enzyme, a broad spectrum solvent, a zwitterionic buffer and at least one primer pair designed by the method above; heating the reaction cocktail at a denaturing temperature of about 90.0xc2x0 C. to about 96.0xc2x0 C. for about 1.0 second to about 30.0 seconds; cooling the reaction cocktail at an annealing/extension temperature of about 50.0xc2x0 C. to about 68.0xc2x0 C. for about 1.0 minute to about 28.0 minutes; repeating the heating and cooling steps at least 10 times; and cooling the reaction cocktail to 4.0xc2x0 C. in a final cooling step.
Other and further objects, features and advantages would be apparent and eventually more readily understood by reading the following specification and by reference to the accompanying drawings forming a part thereof, or any examples of the presently preferred embodiments of the invention given for the purpose of the disclosure.